OncoImmunology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/koni20

Combination checkpoint therapy with anti-PD-1

and anti-BTLA results in a synergistic therapeutic
effect against murine glioblastoma

John Choi, Ravi Medikonda, Laura Saleh, Timothy Kim, Ayush Pant,
Siddhartha Srivastava, Young-Hoon Kim, Christina Jackson, Luqing Tong,
Denis Routkevitch, Christopher Jackson, Dimitrios Mathios, Tianna Zhao,
Hyerim Cho, Henry Brem & Michael Lim

To cite this article: John Choi, Ravi Medikonda, Laura Saleh, Timothy Kim, Ayush Pant,
Siddhartha Srivastava, Young-Hoon Kim, Christina Jackson, Luqing Tong, Denis Routkevitch,
Christopher Jackson, Dimitrios Mathios, Tianna Zhao, Hyerim Cho, Henry Brem & Michael Lim
(2021) Combination checkpoint therapy with anti-PD-1 and anti-BTLA results in a synergistic
therapeutic effect against murine glioblastoma, OncoImmunology, 10:1, 1956142, DOI:
10.1080/2162402X.2021.1956142

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ONCOIMMUNOLOGY
2021, VOL. 10, NO. 1, e1956142 (9 pages)
https://doi.org/10.1080/2162402X.2021.1956142

ORIGINAL RESEARCH

Combination checkpoint therapy with anti-PD-1 and anti-BTLA results in a synergistic

therapeutic effect against murine glioblastoma
John Choia*, Ravi Medikondaa*, Laura Saleha, Timothy Kima, Ayush Panta, Siddhartha Srivastavaa, Young-Hoon Kimb,
Christina Jacksona, Luqing Tonga, Denis Routkevitcha, Christopher Jacksona, Dimitrios Mathios a, Tianna Zhaoa,
Hyerim Choc, Henry Brema, and Michael Lim a
a
Department of Neurosurgery, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, USA; bDepartment of Neurosurgery, College of
Medicine, Asan Medical Center, University of Ulsan, Seoul, Republic of Korea; cDepartment of Radiology, Seoul National University College of
Medicine, Seoul, Republic of Korea

ABSTRACT ARTICLE HISTORY

Clinical trials involving anti-programmed cell death protein-1 (anti-PD-1) failed to demonstrate improved Received 25 January 2021
overall survival in glioblastoma (GBM) patients. This may be due to the expression of alternative check­ Revised 12 July 2021
points such as B- and T- lymphocyte attenuator (BTLA) on several immune cell types including regulatory Accepted 12 July 2021
T cells. Murine GBM models indicate that there is significant upregulation of BTLA in the tumor micro­ KEYWORDS
environment (TME) with associated T cell exhaustion. We investigate the use of antibodies against BTLA glioblastoma
and PD-1 on reversing immunosuppression and increasing long-term survival in a murine GBM model. immunotherapy; anti-PD-1;
C57BL/6 J mice were implanted with the murine glioma cell line GL261 and randomized into 4 arms: (i) anti-BTLA; B and
control, (ii) anti-PD-1, (iii) anti-BTLA, and (iv) anti-PD-1 + anti-BTLA. Kaplan–Meier curves were generated T lymphocyte attenuator;
for all arms. Flow cytometric analysis of blood and brains were done on days 11 and 16 post-tumor immune checkpoint inhibitor
implantation. Tumor-bearing mice treated with a combination of anti-PD-1 and anti-BTLA therapy therapy; glioblastoma
experienced improved overall long-term survival (60%) compared to anti-PD-1 (20%) or anti-BTLA (0%)
alone (P = .003). Compared to monotherapy with anti-PD-1, mice treated with combination therapy also
demonstrated increased expression of CD4+ IFN-γ (P < .0001) and CD8+ IFN-γ (P = .0365), as well as
decreased levels of CD4+ FoxP3+ regulatory T cells on day 16 in the brain (P = .0136). This is the first
preclinical investigation into the effects of combination checkpoint blockade with anti-PD-1 and anti-BTLA
treatment in GBM. We also show a direct effect on activated immune cell populations such as CD4+ and
CD8 + T cells and immunosuppressive regulatory T cells through this combination therapy.

Introduction (TME).8,9 Moreover, regulatory T cells (Tregs) – an immuno­

suppressive T cell population usually not seen in the brain –
Glioblastoma (GBM) is a highly aggressive primary brain
have significantly increased representation within high-grade
tumor with a dismal prognosis.1 Despite current standards of
gliomas such as GBM.10
care involving maximal surgical resection, chemotherapy, and
With anti-PD-1 monotherapy failing to show significant
radiation therapy, median overall survival (mOS) is approxi­
clinical benefit, several studies have examined the efficacy of
mately 19 months.2,3 Recently, the advent of immune check­
combining multiple checkpoint inhibitors in order to target
point blockade (ICB) with antibodies against programmed cell
different pathways of immunosuppression in GBM.11,12 B and
death protein-1 (anti-PD-1) served as a novel immunotherapy
T lymphocyte attenuator (BTLA) is a co-inhibitory checkpoint
treatment strategy that has shown promise in many solid
molecule that is structurally related to PD-1.13 Previous studies
tumors including melanoma and non-small cell lung cancer
have shown that BTLA is highly expressed on tumor-specific
(NSCLC).4,5
T cells in cancer patients14 and is upregulated in several cancers
Given the success of ICB in other cancers, there has been
including gastric cancer,15 hepatocellular carcinoma,16 and
avid interest in applying anti-PD-1 therapy to GBM. However,
GBM.17 Furthermore, it has been shown that increased BTLA
the recent phase III CheckMate 143 clinical trial evaluating
expression correlates with resistance to anti-PD-1 immu­
anti-PD-1 in patients with GBM failed to improve mOS com­
notherapy and poor clinical outcomes.15,16
pared to current standard of care.6,7 The lack of significant
In the context of GBM, the ligand for BTLA – Herpes virus
clinical benefit from anti-PD-1 therapy is thought to be in part
entry mediator (HVEM) – is overexpressed on high-grade
due to both intrinsic resistance to the immune system due to
gliomas and is associated with the suppression of T cell-
poor antigen presentation/priming, low-quality neoantigens,
mediated anti-tumor activity in the GBM TME.18 Given the
and relatively low mutational burden, as well as extrinsic resis­
likely immunosuppressive effect of BTLA in the GBM TME, we
tance from an immunosuppressive tumor microenvironment
sought to evaluate the survival efficacy of anti-BTLA therapy in

CONTACT Michael Lim mklim@stanford.edu Department of Neurosurgery, Johns Hopkins University, Baltimore 21231, USA
*Indicates authors contributed equally to the manuscript
Supplemental data for this article can be accessed on the publisher’s website.
© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
e1956142-2 J. CHOI ET AL.
combination with anti-PD-1 in a murine GBM model and
assess the immunological effects of this dual immune check­
point blockade therapy.
Materials and methods
Murine glioma model and cell lines
6–8 week-old C57BL/6 J wild-type female mice were main­
tained at the Johns Hopkins University Animal Facility per the
Institutional Animal Care and Use Committee (IACUC) pro­
tocol. For all experiments, mice were anesthetized with
Ketathesia (100 mg/kg)/xylazine (10 mg/kg) by intra-
peritoneal (i.p.) injection and had topical eye gel for lubrica­
tion. After every procedure, mice were placed on a heating pad
and observed until fully recovered. Orthotopic murine glioma
models utilized GL261-Luc2 (RRID:CVCL_X986) cells grown
in DMEM (Life Technologies) + 10% FBS (Sigma-Aldrich) +
1% penicillin-streptomycin (Life Technologies) as described in
previous studies (Zeng, 2013). GL261-Luc2 cells were obtained
from Reardon Lab, Dana Farber Cancer Institute. All experi­
ments were performed with mycoplasma-free cells. 1.3e5
GL261-Luc2 cells in a volume of 2 μl were stereotactically
injected 2 mm posterior to the coronal suture, 2 mm lateral
to the sagittal suture, and 3 mm deep to the cortical surface in
the area of the left striatum. After implantation of tumor cells,
mice were assessed for tumor growth on post-implantation day
7 using bioluminescent IVIS imaging (PerkinElmer).
Mice with tumor burden at day 7 were then randomly
separated into control (non-treated) and treatment arms.
Survival experiments were repeated in triplicate with 8–10
mice in each control or treatment arm. Animals were eutha­
nized according to humane endpoints, including central ner­
vous system disturbances, hunched posture, lethargy, weight
loss, and inability to ambulate per our IACUC protocol. Long-
term survivors were rechallenged with re-implantation of
GL261-Luc2 in the right striatum on post-implantation day
60. Additionally, 8 naïve control mice were implanted with
tumor the day of rechallenge to serve as controls.
Therapeutic antibodies
G4 hybridomas were cultured and used to develop hamster
monoclonal antibodies (mAbs) against murine PD-1, as
described in previous studies (Hirano F, 2005). Individual
treatment dose was 200 μg per animal on post-implantation
days 10, 12, and 14 for anti-PD-1 monotherapy and combina­
tion therapy arms. Anti-murine BTLA antibody was received
from Bristol Myers Squibb (BMS) and stored at −80°C in 1 mg/
mL aliquots. Per the manufacturer, anti-BTLA is a non-
depleting, blocking antibody with a defective Fc region pre­
venting antibody-dependent cellular cytotoxicity. This anti­
body was administered in three 400 μg doses on post-
implantation days 7, 10, and 14.
Immune cell harvest and isolation
Mice were deeply anesthetized or euthanized before harvesting
blood (120 μl, retro-orbital) and brains for immunological
assays. 120 μl blood was collected retro-orbitally from mice
under deep anesthesia on days 11 and 16 according to our
IACUC protocol. Red blood cells were lysed using ACK lysis
buffer (ThermoFisher) and resuspended in PBS for further
cytometric analysis. Brains were removed, tissue was mechani­
cally dissociated through a 70 μm filter, and homogenates were
centrifuged (ThermoFisher Sorvall Legend X1R centrifuge) in
a 30%/70% Percoll® (Sigma-Aldrich) gradient at 2200 rpm for
20 minutes without brakes to separate out brain myeloid cells
and lymphocytes from tumor cells and myelin. Brain immune
cells were extracted at the 30%/70% interface and resuspended
in phosphate-buffered saline (PBS) buffer (Sigma-Aldrich) for
further cytometric analysis.
Flow cytometric analysis of murine immune cells
Mouse immune cells were stained for Live/Dead (L/D)
(Invitrogen), CD45, CD3, CD4, CD8, PD-1, IFN-γ, FoxP3,
and BTLA (Supplementary Figure S1 for antibody fluoro­
phores). To stain for the intracellular marker IFN-γ and intra­
nuclear marker FoxP3, samples were fixed in 1:3 fixation/
permeabilization concentrate:diluent mixture (eBioscence) for
30 minutes and subsequently stained in permeabilization buf­
fer (eBioscience). Fluorescence minus one (FMO) was used to
control for data spread due to multiple fluorochromes and
nonbinary expression of markers such as IFN-γ. Flow data
was acquired using a FACSCelesta flow cytometer (BD) and
analyzed using FlowJo (BD). Nonviable cells and doublets were
excluded by forward versus side scatter gating, forward scatter
height versus forward scatter area gating, and L/D staining.
Co-culture and ELISA
For IFN-γ assays, 5e3 CD45.2+ CD3+ CD8 + T cells were
sorted from each mouse brain and co-cultured with 100 μg/
ml GL261-Luc2 tumor cell lysate and 25e3 dendritic cells iso­
lated from CD45.1 mouse spleen (isolated with a pan-dendritic
cell isolation kit) (Miltenyi Biotec) in a 96-well round bot­
tomed plate with T cell media (RPMI 1640 + 10% FBS + 1%
NEAA + 1% 2-Mercaptoethanol + 1% Penicillin/
Streptomycin). Co-cultured cells were incubated at 37°C for
48 hours. Supernatant was collected for subsequent ELISA for
IFN-γ and run on a plate reader (PerkinElmer Victor3 1420
Multilabel plate reader).
Depletion study
GL261-luc2 tumor was confirmed in mice on post-
implantation day 7. After randomization, tumor bearing mice
received IP injections of either anti-CD4 (clone GK1.5, Bio
X Cell, catalog#: BE0003-1) at 200 μg/dose or anti-CD8
(clone 2.43, Bio X Cell, catalog#: BE0004-1) at 500 μg/dose.
Control mice received 200 μL of PBS IP. All depletion anti­
bodies were administered 48 hours and 24 hours prior to the
administration of the first dose of anti-BTLA treatment.
Depletion antibodies were administered every seven days
thereafter until conclusion of the study. T cell depletion was
confirmed via flow cytometry of blood in each treatment arm
on day 14 post-implantation.

Statistical analysis
All replicates were biological replicates. Survival was analyzed
via Kaplan-Meier method and compared by log-rank (Mantel-
Cox) test. Calculated variables were treated as continuous
variables under the assumption that data follow Student
T-distribution. Mouse experimental data were analyzed using
a 2-tailed Student’s T-test for experiments containing 2 groups.
All data were analyzed using GraphPad Prism 8 and values of
P < .05 were considered statistically significant.
Results
BTLA expression increases over time on tumor-infiltrating
non-Treg CD4 + T cells
To evaluate the expression of BTLA over time on immune cells,
a time point experiment was conducted in mice on post-
implantation day 7, 16, and 21 (Figure 1). 1.3e5 GL261-Luc+
cells were implanted in the left striatum on day 0. Blood and
brain were harvested at each of the three time points for 5 mice
after confirming presence of tumor on IVIS. Single cell suspen­
sions were prepared and stained for flow cytometric analysis of
BTLA. In the blood, BTLA expression significantly increased
on non-Treg CD4 + T cells from 6.1% on day 7 to 55.1% on day
16 (P < .0001) and 67.8% on day 21 (P < .0001) (Figure 1b). In
the brain, BTLA expression on tumor-infiltrating Treg (CD3
+ CD4+ FoxP3+) cells did not significantly change from an
average of 13.4% on day 7 to 16.2% on day 16 (P = .003) and
20.0% on day 21 (P = .003) (Figure 1c). However, BTLA
expression on tumor-infiltrating non-Treg (CD3+ CD4
+ FoxP3-) T cells significantly increased from a mean of
10.1% on day 7 to 37.7% on day 16 (P < . 0001) and 37.8%
on day 21 (P < .0001) (Figure 1c). On CD3+ CD8 + T cells in
the blood, BTLA expression increased from 8.2% on day 7 to
35.3% on day 21 (P < .001) (Figure 1b). In the brain, BTLA
Figure 1. Timing of BTLA expression in tumor-bearing mice on days 7, 16, and 21. (a) Representative flow cytometry plots of TILs demonstrating BTLA expression
on day 7, 16, and 21 on CD4+ FoxP3 + T cells, CD4+ FoxP3- T cells, and CD8 + T cells (b)Aggregate (n = 5) flow cytometry data of BTLA expression in PBMCs from
untreated tumor-bearing mice on days 7, 16, and 21. (c) Aggregate (n = 5) flow cytometry data of BTLA expression in TILs of untreated tumor-bearing mice on days 7,
16, and 21. All plots represent mean with standard deviation. Comparison between two groups is made via the Student’s t test. (* p < .05).
ONCOIMMUNOLOGY e1956142-3
expression on CD3+ CD8 + T cells did not significantly change
from 12.9% on day 7 to 17.1% on day 21 (P = .49) (Figure 1c).
Next, expression of BTLA and its ligand HVEM was studied
in the glioma setting on various tumor-infiltrating immune cell
populations including CD45+ CD3 + T cells, CD45+ CD11b+
cells (commonly denoting macrophages), CD45+ CD11 c
+ cells (commonly denoting macrophages and dendritic cells)
and CD45+ CD19 + B cells (Supplemental Figure S1). There
was significantly lower HVEM expression on CD3+ TILs com­
pared to tumor-infiltrating CD11b+ cells (P < .0001), CD11 c
+ cells (P < .0001), or CD19 + B cells (P < .0001) (Supplemental
Figure S1b). There was significantly lower BTLA expression on
CD11 c+ cells compared to CD3 + T cells (P = .0405) and
tumor-infiltrating CD11b+ cells (P = .0062) (Supplemental
Figure S1b). HVEM expression was also analyzed after admin­
istration of control (no treatment), anti-PD-1 alone, anti-BTLA
alone or combination therapy. There was no significant differ­
ence in HVEM expression on tumor-infiltrating CD11b+ cells,
CD11 c+ cells, or CD19 + B cells with the four treatment arms.
There was significantly higher HVEM expression on CD3 + T
cells with anti-BTLA (P = .0135) or combination therapy
(P = .0285) compared to control (Supplemental Figure S1c).
Combination anti-BTLA and anti-PD-1 treatment increases
activation of CD4+ and CD8 + T cells and modulates presence of
Tregs in the brain and blood
In order to assess the immunologic effects of anti-PD-1 and
anti-BTLA in murine GBM models, blood was harvested on
days 11 and 16 with brains also harvested on day 16. T cells
were gated on forward versus side scatter for lymphocyte
populations with exclusion of doublets, which were then
gated for live CD45+ CD3 + T cells and separated into CD4
+ and CD8+ populations (Supplemental Figure S2).
On day 16 in the brain, IFN-γ expression of CD4+ and CD8
+ populations were compared between experimental arms,
with 32.9% of CD4+ tumor-infiltrating lymphocytes (TILs)
expressing IFN-γ in the combination anti-PD-1 and anti-
e1956142-4 J. CHOI ET AL.
BTLA arm compared to 11.7% in the non-treated (control)
arm, 16.7% in the anti-PD-1 monotherapy arm (P < .0001), and
19.1% in the anti-BTLA arm (P = .0033) (Figure 2a, b).
Similarly, 76.3% of CD8+ TILs expressed IFN-γ in the combi­
nation anti-PD-1 and anti-BTLA arm compared to 43.8%,
59.9%, and 44.8% in control, anti-PD-1 monotherapy
(P = .0365), and anti-BTLA (P = .0004) monotherapy arms,
respectively (Figure 2b).
Of note, in the combination therapy arm, there was
a marked decrease in the proportion of CD4+ FoxP3 + T regs
of the parent CD4+ TIL population in the brain compared to
monotherapy and control arms, with 11.7% of CD4+ TILs
expressing FoxP3 in mice treated with both anti-PD-1 and
anti-BTLA compared to 36.0% of CD4 + T cells in non-
treated mice expressing FoxP3 and 27.9% of CD4+ TILs in
mice treated with anti-PD-1 monotherapy expressing FoxP3
(P = .0136) (Figure 2b). However, there was no statistically
significant difference between combination and anti-BTLA
monotherapy arms for FoxP3 expression in the brain, with
15.8% of CD4+ TILs in mice with anti-BTLA monotherapy
expressing FoxP3 (P = .2349). However, there was a statistically
significant difference in FoxP3 expression between anti-PD-1
and anti-BTLA monotherapy arms (P = .0483) (Figure 2a, b).
Next, we evaluated for the expression of additional co-
stimulatory molecules such as 4–1BB and co-inhibitory mole­
cules such as TIM-3 and LAG-3 on TILs. 4–1BB expression on
CD4+ TILs was significantly higher with combination therapy
(31.6%) compared to control (23. 2%, P = .0098), anti-PD-1
monotherapy (23.7%, P = .0135), anti-BTLA monotherapy
(19.1%, P = .0007) (Figure 2c). Likewise, 4–1BB expression
on CD8+ TILs was significantly higher with combination
Figure 2. Flow cytometric analysis of T cell populations in mouse brain at day 16 post-tumor implantation. (a) Flow plots demonstrating CD4 and CD8 IFN-γ
expression, 4–1BB expression, and CD4 Foxp3 presence in the brain. (b) Aggregate (n = 5 per group) data of control (no treatment), anti-PD-1, anti-BTLA, and
combination therapy groups for CD4 and CD8 IFN-γ and CD4 Foxp3 presence in the brain. Differences between two treatment arms were analyzed via Student’s t test.
(**** P < .0001, *** P < .001, ** P < .01, * P < .05).
therapy (38.7%) compared to control (29.0%, P = .016), anti-
PD-1 monotherapy (21.2%, P = .0016), anti-BTLA monother­
apy (18.1%, P = .0063) (Figure 2c). There was no significant
difference in LAG-3 or TIM-3 expression between control,
monotherapy, or combination therapy.
We also studied if anti-PD-1 and anti-BTLA combination
therapy was affecting tumor-infiltrating myeloid or B cell
populations. After treating mice with control, anti-PD-1 only,
anti-BTLA only, or combination therapy, we found no signifi­
cant change in tumor infiltration of CD45+ CD11b+ macro­
phages between the four treatment arms. Of note, there was
a trend toward significantly greater CD45+ CD11c+ macro­
phage and dendritic cell infiltration of the tumor in the com­
bination therapy arm compared to control (P = .09). Likewise,
there was a trend toward significantly greater CD45+ CD19 + B
cell tumor infiltration with combination therapy compared to
control (P = .06) (Supplemental Figure S3).
To assess the characteristics of possible homing and periph­
eral proliferation of lymphocytes, blood was analyzed on day
11, one day after exposure to both anti-PD-1 and anti-BTLA, as
well as on day 16 – the day of brain harvest. Indications of
T cell homing were present across all arms for CD8 + T cells,
with marked decrease in percentage of CD8+ IFN-γ-secreting
T cells on day 16 compared to day 11. Furthermore, combina­
tion treatment with anti-PD-1 and anti-BTLA showed the
greatest levels of CD4+ and CD8+ IFN-γ expression on day
11 with 21.5% of CD4+ cells and 37.9% of CD8+ cells expres­
sing IFN-γ (Supplemental Figure S4). Notably, tumor bearing
mice treated with combination therapy did not observe
a similar trend in peripheral regulatory T cell distribution on
days 11 and 16; while other monotherapy arms showed

a marked decrease in Tregs between days 11 and 16, mice
treated with combination therapy showed similar proportions
of Tregs in the blood on both days (1.52% and 1.56% on days
11 and 16, respectively) (P = .8271) (Figure S3).
Treatment with anti-BTLA and anti-PD-1 correlate with
increased IFN-γ secretion
To further determine if combination therapy with anti-PD
-1 and anti-BTLA resulted in decreased immunosuppression,
TILs belonging to mice ± treatment with anti-PD-1 monother­
apy, anti-BTLA monotherapy, or combination therapy were
sorted for live CD45+ CD3+ CD8 + T cells and co-incubated
with dendritic cells and GL261-Luc2 tumor cell lysate
(Figure 3a). Resultant supernatant with secreted IFN-γ was
then analyzed by ELISA. IFN-γ levels directly correlated with
treatment, with significantly increased secretion of IFN-γ in
combination treatment compared to no treatment (P = .0033)
and anti-BTLA monotherapy (P = .0032). There is a trend
toward increased secretion of IFN-γ in combination treatment
compared to anti-PD-1 monotherapy (P = .0776) (Figure 3b).
Combination treatment with anti-PD-1 and anti-BTLA
results in improved overall long-term survival
Having confirmed BTLA expression on Tregs in GBM, we
hypothesized that dual blockade with anti-PD-1 and anti-
BTLA antibodies would result in a synergistic survival benefit
due to targeted reversal of immunosuppression of CD8+ and
CD4 + T cells. After ensuring tumor burden with GL261-Luc2
on post-implantation day 7, mice were treated with three doses
of BTLA on days 7, 10, and 14 as well as anti-PD-1 on days 10,
12, and 14 per previous protocols19 (Figure 4a, b). Compared to
anti-PD-1 monotherapy, combination treatment with dual
checkpoint blockade resulted in increased overall survival
(60% vs. 20%, P = .0003). There were no long-term survivors
with anti-BTLA monotherapy as well as no statistically signifi­
cant difference between control and anti-BTLA monotherapy
for median overall survival (P = .0954). On post-implantation
day 60, long-term survivors and naïve control mice were
Figure 3. In vitro co-culture assay for IFN-γ secretion with ELISA. (a) CD8 + T cells were isolated via FACS microfluidic sorting from untreated mice and mice treated
in vivo with anti-PD-1 monotherapy, anti-BTLA monotherapy, and combination anti-PD-1 and anti-BTLA. Cells were co-cultured with GL261 tumor cell lysate and CD11c
+ dendritic cells. (b) Plot of concentration of IFN-γ in supernatant of co-culture wells. All plots represent mean with standard error. Differences between two treatment
arms were analyzed via Student’s t test. (**** P < .0001, *** P < .001, ** P < .01, * P < .05).
ONCOIMMUNOLOGY e1956142-5
contralaterally rechallenged/implanted with intracranial
GL261-Luc2. Long-term survivors rechallenged with glioma
demonstrated significantly better survival than control mice
(P = .0031) (Supplemental Figure S5).
Depletion of CD4 + T cells or CD8 + T cells diminishes the
efficacy of anti-PD-1 and anti-BTLA combination therapy
The results thus far suggest that anti-PD-1 and anti-BTLA
combination therapy provide a synergistic survival benefit in
murine glioma (Figure 4) potentially mediated through CD4
+ and CD8 + T cells (Figures 2, Figures 3). To determine whether
CD4+ and CD8 + T cells are necessary for the observed survival
benefit with combination therapy, we performed a depletion
study (Figure 5). After confirming tumor burden on post-
implantation day 7, mice were depleted of either CD4 + T cells
or CD8 + T cells. After depletion of these T cell populations,
mice were treated with anti-PD-1 plus anti-BTLA combination
therapy. Depletion of CD4+ or CD8 + T cells was confirmed on
Day 14 (Supplemental Figure S6). There was no significant
difference in survival between the control arm, CD4 + T cell
depletion plus combination therapy arm, and the CD8 + T cell
depletion plus combination therapy arm. Combination therapy
without any T cell depletion had significantly greater median
overall survival compared to control (P = .004), CD4 + T cell
depletion plus combination therapy (P = .036), and CD8 + T cell
depletion plus combination therapy (P = .046).
Discussion
Despite advances in immune checkpoint blockade for solid
tumors, GBM has demonstrated resistance to anti-PD-1 ther­
apy with multiple mechanisms of immunosuppression.9 Of
note, beyond PD-1, TILs in GBM demonstrate upregulation
of additional inhibitory immune checkpoint molecules such as
Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4),
Indoleamine 2,3-dioxygenase 1 (IDO1), T-cell immunoglobu­
lin and mucin-domain containing-3 (TIM-3), and
Lymphocyte-activation gene-3 (LAG-3).17 As such, the utiliza­
tion of combination checkpoint therapy involving additional
e1956142-6 J. CHOI ET AL.

Figure 4. Survival schema and Kaplan-Meier curve. (a) Mice were implanted with 1.3e5 GL261-Luc2 cells with a stereotactic frame. On post-implantation day 7, mice
were assessed for presence of tumor by injecting them with 200 μl of 1 mg/ml D-luciferin and imaged with IVIS (In Vivo Imaging Systems), and subsequently randomly
assorted into four groups: control (no treatment), anti-BTLA monotherapy, anti-PD-1 monotherapy, and combination anti-BTLA and anti-PD-1 therapy. 5 mice in each
arm were saved for blood and brain harvest to undergo flow cytometric analysis. Remaining 8 mice in each arm underwent a survival study and were rechallenged
on day 60. (b) Treatment with anti-BTLA was on days 7, 10, and 14. Treatment with anti-PD-1 was on days 10, 12, and 14. (c) Kaplan-Meier curve demonstrating
differences in overall long-term survival with combination therapy. (**** P < .0001, *** P < .001, ** P < .01, * P < .05).

checkpoint blockade such as antibodies against CTLA-4, LAG-
3, TIM-3, and IDO1 have been explored in both pre-clinical
and clinical investigations.
In the clinical setting, the precedent for success with com­
bination checkpoint therapy for brain tumors is most salient in
treatment for metastatic melanoma, in which anti-CTLA-4 and
anti-PD-1 combination therapy was approved by the FDA in
2016 as a first-line therapy and showed promising results of
improved progression-free survival (PFS) compared with anti-
PD-1 therapy alone.20 There is currently an ongoing trial that is
studying antibodies against PD-1 and LAG-3 (NCT02658981),
with several other combination studies bolstered by promising
preclinical findings.11,12,21In preclinical GBM murine models,
combination therapy involving antibodies against TIM-3 (anti-
TIM-3), anti-PD-1, and stereotactic radiation resulted in 100%
overall survival.11 As the success of this mechanism is thought
to be in part from targeting both myeloid and lymphocyte
populations with anti-TIM-3 and anti-PD-1, respectively,
these preclinical studies show proof of principle of the syner­
gistic survival benefit that can result from targeting multiple
pathways of immunosuppression.
In this study, we found a synergistic survival benefit accom­
panying combination therapy with anti-PD-1 and anti-BTLA
in a mouse glioma model, with increased 4–1BB expression
and IFN-γ secretion from CD4+ and CD8 + T cells. There was
also a decreased proportion of infiltrating Tregs of the CD4
+ TILs (Figures 2, Figures 4c). In tumor-bearing mice, combi­
nation therapy demonstrated T cells with significantly
increased IFN-γ expression in both CD4+ and CD8 + T cell
populations that correlated with survival outcomes. This rever­
sal of immunosuppression when compared to control and
monotherapy arms in conjunction with a decreased proportion
of Treg infiltration demonstrates that BTLA therapy may pos­
sibly involve a CD4-based mechanism, likely involving

Figure 5. CD4+ and CD8 + T cell depletion Kaplan-Meier curve. Mice in the depletion arms underwent depletion of either CD4 + T cells or CD8 + T cells prior to
treatment with anti-BTLA and anti-PD-1 therapy. There was no significant difference in survival between the control arm, CD4 + T cell depletion arm, and CD8 + T cell
depletion arm. Combination therapy without any T cell depletion had significantly greater median overall survival compared to control (P = .004), CD4 + T cell depletion
plus combination therapy (P = .036), and CD8 + T cell depletion plus combination therapy (P = .046).
decreased polarization of CD4+ TILs toward the Treg pheno­
type in the TME, leading to a greater proportion of non-Treg
effector CD4+ TILs (Figure 2a, b, 3b). Furthermore, our deple­
tion study (Figure 4) suggests that both CD4 + T cells and
CD8 + T cells are necessary for the synergistic survival benefit
observed with anti-PD-1 and anti-BTLA combination therapy
(Figure 5).
CD4+ FoxP3+ Treg cells in the TME of GBM are
thought to play an important role in immunosuppression.
Both preclinical and clinical studies have shown increased
infiltration of Tregs within GBM,22–24 with data showing
that the presence of these increased Treg cell fractions in
patients correlates with decreased effector T cell prolifera­
tion and responsiveness.25 Several studies have since
explored the potential survival impact of Treg based ther­
apy in GBM,26 with anti-CD25 or anti-CTLA demonstrat­
ing preclinical efficacy by targeting Tregs.22,27,28 However,
these therapies have not yet successfully been translated
into the clinical setting.29
Interestingly, our BTLA timepoint experiment (Figure 1)
demonstrated differential expression of BTLA on tumor-
infiltrating CD4+ FoxP3+ Treg cells and CD4+ FoxP3- non-
Treg cells over time. BTLA expression remained constant over
time on tumor-infiltrating Treg cells, however BTLA expression
significantly increased over time on tumor-infiltrating non-Treg
cells. Indeed, there is a similar percentage of BTLA expression on
both tumor-infiltrating Treg cells and tumor-infiltrating non-
Treg cells on day 7 (13.4% vs. 10.1% respectively). However,
there is significantly higher BTLA expression on tumor-
infiltrating non-Treg cells compared to tumor-infiltrating Treg
cells by day 21 (37.8% vs 20.0%). This data parallels data in
human GBM patients showing PD-1 expression on TILs is
increased in patients with GBM compared to control.30
Given that BTLA is an immune checkpoint, we posit that
tumor-infiltrating non-Treg cells become more suppressed over
time due to increased BTLA expression over the course of tumor
progression. We believe there may not be BTLA-mediated sup­
pression of tumor-infiltrating Treg cells given constant low
expression of the BTLA immune checkpoint on this cell
ONCOIMMUNOLOGY e1956142-7
population over tumor progression from day 7 to day 21. Our
data also showed that there was not a statistically significant
difference in the infiltration of Tregs between anti-BTLA and
combination therapy arms. This has two possible implications:
(1) the immune phenomenon affecting Treg infiltrating with
combination therapy is likely due to the inhibition of BTLA
rather than PD-1 and (2) despite a similar decrease in Treg
infiltration, CD8+ IFN-γ expression as well as survival outcomes
with anti-BTLA monotherapy failed to reach the same level as
anti-PD-1 monotherapy or combination therapy (Figures 2b,
Figures 4). Interestingly, while the immunosuppressive role of
Tregs have been well explored in both preclinical models and in
human GBM tissue, the impact of Tregs on overall survival and
prognosis are less clear. An immunohistochemical analysis of
glioma tissue by Heimberger et al. with multivariate analysis for
the presence of Tregs on overall survival found that when
accounting for confounding factors such as age and Karnofsky
performance score, the presence of FoxP3+ Tregs did not have
a significant prognostic impact.10 This may in part offer insights
as to why anti-BTLA monotherapy may not confer as great
a survival benefit, but in turn is an excellent candidate for
combination immunotherapy as targeting Tregs has been
shown to impact immunosuppression in GBM.10
Additional lines of exploration that would be essential in
characterizing this avenue of treatment would be to delve into
further mechanisms involving BTLA, including understanding of
involved essential trafficking molecules since our data suggests
anti-BTLA decreases Treg infiltration into the brain. For example,
a notable target that has already been shown to be highly asso­
ciated with Treg trafficking in GBM is C-C chemokine receptor
type 4 (CCR4), which is highly expressed on Tregs with its cognate
antigen being produced by GBM.31 Furthermore, our supplemen­
tal data suggests BTLA and HVEM are also expressed on myeloid
and B cells. In our study, we did not find a significant change in
tumor-infiltrating CD11b+ cells, CD11c+ cells, or CD19 + B cells
with combination therapy compared to control. Despite this
result, It may be interesting to characterize in future studies
whether anti-BTLA therapy affects the interaction between mye­
loid or B cells and TILs.
e1956142-8 J. CHOI ET AL.
With any animal model research, consideration must be
given to the degree to which the animal model accurately
mimics human disease. GBM is notorious for being difficult
to replicate in vivo. Indeed, many therapeutic combinations
that work well in murine glioma models fail to translate to
clinical trials in human GBM.6,19,32 In this project, all
experiments were conducted using the GL261-Luc2 cell
line. The GL261 cell line is one of the most well-studied
glioma models in the field of GBM immunotherapy.33 The
primary benefit of this cell line is that it is well character­
ized, can be easily implanted into immunocompetent
C57BL/6 mice given that it is syngeneic, and effectively
recapitulates many of the characteristics of human
GBM.34,35 The Luc2 tag allows for tumor visualization on
IVIS, and indeed many recent studies evaluating checkpoint
inhibition therapy in GBM have used the GL261-Luc2
model.11,19,32,36 The primary disadvantage of this model is
that it does not fully recapitulate the significant intra-
tumoral and inter-tumoral heterogeneity of GBM.
Furthermore, the GL261-Luc2 model is a moderately
immunogenic model whereas GBM is significantly
immunoresistant.35 It is important to bear in mind the
strengths and limitations of the preclinical model utilized
to study GBM in the preclinical setting, and additional
experiments should be conducted in other GBM cell lines
to improve generalizability of these results.
The appeal of BTLA as a target for immunotherapy in
GBM comes from its multimodal effect on regulatory
T cells and CD8 + T cells. The dramatic changes in Treg
representation as well as the synergistic survival benefit
seen in murine in vivo models demonstrate a treatment
schema that can target multiple immunosuppressive cells
when used in combination with antibodies involved in the
well-established PD-1 pathway. With immune checkpoint
therapy continuing to gain traction in the clinical setting,
new targets that synergize survival outcomes are of interest.
Our preclinical study involving antibodies against PD-1 and
BTLA offer proof of principle for the efficacy of this com­
bination therapy and offers the first exploration of targeting
BTLA in GBM in a mouse model.
Abbreviations
Band T lymphocyte attenuator (BTLA); Bristol Myers Squibb (BMS);
C-C chemokine receptor type 4 (CCR4); Cytotoxic T-lymphocyte-
associated protein 4 (CTLA-4); Fluorescence minus one (FMO);
Glioblastoma (GBM); Herpes virus entry mediator (HVEM); immune
checkpoint blockade (ICB); Indoleamine 2,3-dioxygenase 1 (IDO1);
Institutional Animal Care and Use Committee (IACUC); intra-
peritoneal (i.p.); IVIS (In Vivo Imaging System); Lymphocyte-activation
gene-3 (LAG-3); median overall survival (mOS); monoclonal antibodies
(mAbs); non-small cell lung cancer (NSCLC); phosphate-buffered saline
(PBS); programmed cell death protein-1 (anti-PD-1); progression-free
survival (PFS); regulatory T cells (Tregs); T-cell immunoglobulin and
mucin-domain containing-3 (TIM-3); tumor infiltrating lymphocytes
(TILs); tumor microenvironment (TME);
Novelty and Impact:
BTLA is an immune checkpoint thought to downregulate the immune
system. BTLA has been shown to be upregulated in the GBM tumor
microenvironment. In this study, we study synergy between anti-BTLA
and anti-PD-1 immunotherapy and show that combination therapy sig­
nificantly improves survival in a murine GBM model. Furthermore, we
demonstrate a direct effect of combination therapy on several immune cell
populations including CD4+, CD8+, and regulatory T cells.
Disclosure statement
The authors do not have any relevant conflicts of interest associated with
this manuscript.
Funding
This research was funded from a gift fund by private donors Conflict of
Interest: Michael Lim (Funding from Arbor Pharmaceuticals, Accuray,
BMS, Novartis; Consultant: BMS, Merck, SQZ Biotechnologies, Tocagen,
VBI; Patents: Combining Focused Radiation and Immunotherapy,
Combining Local Chemotherapy and Immunotherapy)
ORCID
Dimitrios Mathios http://orcid.org/0000-0002-0792-6880
Michael Lim http://orcid.org/0000-0003-0523-3076
Data availability statement:
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Ethics statement:
This study protocol was approved by the Johns Hopkins Institutional
Animal Care and Use Committee. This study did not involve any
human patients.
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